CA2599477A1 - Methods and compositions for improving fidelity in a nucleic acid synthesis reaction - Google Patents

Methods and compositions for improving fidelity in a nucleic acid synthesis reaction Download PDF

Info

Publication number
CA2599477A1
CA2599477A1 CA002599477A CA2599477A CA2599477A1 CA 2599477 A1 CA2599477 A1 CA 2599477A1 CA 002599477 A CA002599477 A CA 002599477A CA 2599477 A CA2599477 A CA 2599477A CA 2599477 A1 CA2599477 A1 CA 2599477A1
Authority
CA
Canada
Prior art keywords
nucleotide
triphosphate
derivative
nucleic acid
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002599477A
Other languages
French (fr)
Inventor
Philip Richard Buzby
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helicos BioSciences Corp
Original Assignee
Helicos Biosciences Corporation
Philip Richard Buzby
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Helicos Biosciences Corporation, Philip Richard Buzby filed Critical Helicos Biosciences Corporation
Publication of CA2599477A1 publication Critical patent/CA2599477A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/34Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing

Abstract

The invention provides methods and compositions for improving the fidelity of a sequencing-by-synthesis reaction by using a nucleotide derivative that forms a hydrogen bond with a complementary nucleotide on a template, but fails to form a phosphodiester bond with the 3' hydroxyl group of a primer under conditions otherwise suitable for a polymerization reaction; thereby blocking incorporation of a mismatched nucleotide.

Description

METHODS AND COMPOSITIONS FOR IMPROVING FIDELITY
IN A NUCLEIC ACID SYNTHESIS REACTION

Technical Field of the Invention [0001] The invention generally relates to methods for improving fidelity in a nucleic acid synthesis reaction, and more particularly to methods for sequencing a nucleic acid using a nucleotide derivative that decreases the rate of misincorporation of nucleotides in a nucleic acid synthesis reaction.

Back2round of the Invention [0002] In vitro nucleic acid synthesis is a foundation of many fundamental research and diagnostic tools, such as nucleic acid amplification and sequencing. In a template-dependent nucleic acid synthesis reaction, the sequential addition of nucleotides is catalyzed by a nucleic acid polymerase. Depending on the template and the nature of the reaction, the nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, or a reverse transcriptase. DNA
synthesis requires a 3' hydroxyl on the primer for addition at the a-phosphate of a free deoxynucleotide triphosphate (dNTP). A phosphodiester bond between the nucleotide on the 3' terminus of the primer and the free nucleotide is formed and diphosphate is released. This reaction is shown schematically in Figure 1.
[0003] The fidelity of template-dependent nucleic acid synthesis depends in part on the ability of the polymerase to discriminate between complementary and non-complementary nucleotides. Normally, the conformation of the polymerase enzyme favors incorporation of the complementary nucleotide. However, there is still an identifiable rate of misincorporation that depends upon factors such as local sequence and the base to be incorporated.
[0004] Synthetic or modified nucleotides and analogs, such as labeled nucleotides, tend to be incorporated into a primer less efficiently than naturally-occurring dNTPs. The reduced efficiency with which the unconventional nucleotides are incorporated by the polymerase can adversely affect the performance of sequencing techniques that depend upon faithful incorporation of such unconventional nucleotides.
[0005] Single molecule sequencing techniques allow the evaluation of individual nucleic acid molecules in order to identify changes and/or differences affecting genomic function. In single molecule techniques, individual, optically-resolvable nucleic acid fragments are attached to a solid support, and sequencing is conducted on the individual strands.
Sequencing events are detected and correlated to the individual strands. See Braslavsky et al., Proc. Natl. Acad. Sci., 100: 3960-64 (2003), incorporated by reference herein. Because single molecule techniques do not rely on ensemble averaging as do bulk techniques, errors due to misincorporation can have a significant deleterious effect on the sequencing results. The incorporation of a nucleotide that is incorrectly paired, under standard Watson and Crick base-pairing, with a corresponding template nucleotide during primer extension may result in sequencing errors. The presence of misincorporated nucleotides may also result in prematurely terminated strand synthesis, reducing the number of template strands for future rounds of synthesis, and thus reducing the efficiency of sequencing.
[0006] There is, therefore, a need in the art for improved methods for reducing the frequency of misincorporation and improving fidelity in nucleic acid synthesis reactions, especially in single molecule sequencing.

Summary of the Invention [0007] The invention addresses the problem of misincorporations in nucleic acid synthesis reactions. The invention improves the fidelity of nucleic acid synthesis reactions by selectively blocking incorporation of non-complementary nucleotides in a template-dependent sequencing-by-synthesis reaction. In order to block misincorporation, nucleotide derivatives that hydrogen bond to their complement on the template, but do not engage in phosphodiester bonding with adjacent primer nucleotides, are added to the reaction along with labeled standard nucleotides for incorporation into the primer. A standard (labeled) nucleotide typically will out-compete derivatives for complementary binding. In cases in which a derivative attaches at an incorporation site intended for the complementary standard nucleotide, the derivative will be washed out and the position will be occupied by a standard nucleotide in a subsequent round.
Because a complementary derivative will typically out-compete a mismatched standard nucleotide for incorporation, the derivatives end up blocking misincorporation. Derivatives, which are only hydrogen-bonded to their complement, are labile in comparison to standard nucleotides, which engage in phosphodiester bonding to adjacent primer nucleotides (in addition to complement hydrogen bonding). Thus derivatives can be washed out, making their complement available for binding in subsequent nucleotide addition cycle.
[0008] Methods of the invention prevent some or all of the misincorporation errors that result in erroneous base calling and/or chain termination in sequencing reactions. According to the invention, a polymerization reaction is conducted on a nucleic acid duplex that comprises a primer hybridized to a template nucleic acid. The reaction is conducted in the presence of a polymerase, at least one free dNTP corresponding to a first nucleotide species, and at least one -nucleotide derivative corresponding to a nucleotide species different from the dNTP. The nucleotide derivative comprises a modification that inhibits the formation of a phosphodiester bond between the derivative and the free 3' hydroxyl group of the terminal nucleotide on the primer. As such, the nucleotide derivative cannot incorporate into the primer.
However, if the template comprises a nucleotide at a position immediately adjacent to the 3' terminus of the primer that is complementary to the nucleotide derivative, the nucleotide derivative forms hydrogen bonds with its complementary nucleotide and prevents the incorporation of the free nucleotide into the primer. According to the invention, derivatives that are complementary to an available template nucleotide out-compete misincorporating dNTPs for template binding.
[0009] In a single molecule sequencing-by-synthesis reaction, primer/template duplexes are bound to a solid support such that each duplex is individually optically detectable.
According to the invention, a primer/template duplex is exposed to a polymerase, a labeled nucleotide corresponding to at least a first nucleotide species, and at least one nucleotide derivative corresponding to a nucleotide species different than the labeled species. The duplex may be simultaneously exposed to the polymerase, labeled nucleotide and nucleotide derivative;
or it may be first exposed to the nucleotide derivative and then to the polymerase and labeled nucleotide. In a preferred aspect, the duplex is simultaneously exposed to the polymerase, the free nucleotide and three nucleotide derivatives, one corresponding to each of the three remaining nucleotide species. Unincorporated labeled nucleotides are washed away, and because hydrogen bonds are relatively weak, the nucleotide derivatives are removed along with the unincorporated labeled nucleotides. The incorporation of the labeled nucleotide is determined, as well the identity of the nucleotide that is complementary to a nucleotide on the template at a position that is opposite the incorporated nucleotide. The polymerization reaction is serially repeated in the presence of labeled nucleotide that corresponds to each of the other nucleotide species, and appropriate nucleotide derivatives, until a sequence of incorporated nucleotides is compiled from which the sequence of the template nucleic acid can be determined.
[0010] Single molecule sequencing methods of the invention preferably comprise template/primer duplex attached to a surface. Individual nucleotides added to the surface comprise a detectable label - preferably a fluorescent label. Each nucleotide species can comprise a different label, or they can comprise the same label. In a preferred embodiment, each duplex is individually optically resolvable in order to facilitate single molecule sequence discrimination. The choice of a surface for attachment of duplex depends upon the detection method employed. Preferred surfaces for methods of the invention include epoxide surfaces and polyelectrolyte multilayer surfaces, such as those described in Braslavsky, et al., supra. Surfaces preferably are deposited on a substrate that is amenable to optical detection of the surface chemistry, such as glass or silica. The precise surface and substrate used in methods of the invention is immaterial to the functioning of the invention.
[0011] The invention provides also compositions for use in a nucleic acid synthesis reaction. In one embodiment, the compositions comprise labeled nucleotide corresponding to a first nucleotide species and at least one nucleotide derivative corresponding to a different nucleotide sequence. Methods and compositions of the invention may take into account that different nucleotides incorporate in an extending primer at different rates and that misincorporation rates similarly vary with the type of nucleotide being incorporated.
Accordingly, the identity of the one or more nucleotide derivatives used in methods and compositions of the invention can be varied with the identity of the correct nucleotide sought to be incorporated. For exaniple, if guanidine triphosphate frequently misincorporates at an incorporation site in which adenosine triphosphate should be incorporated, but never misincorporates at an incorporation site in which thymidine triphosphate or cytidine triphosphate should be incorporated, there is no need to add nucleotide derivatives corresponding to thymidine triphosphate or cytidine triphosphate in a synthesis reaction in which guanidine triphosphate is used as the free nucleotide.
[0012] According to the invention, a nucleotide derivative forms a hydrogen bond with a complementary nucleotide on a template nucleic acid, but comprises a modification that prohibits the polymerase from incorporating the derivative into the primer because it prevents the forination of a phosphodiester bond with the 3' hydroxyl group of the primer.
Preferred nucleotide derivatives comprise a modification to the normal nucleotide triphosphate group to prevent formation of a phosphodiester bond. Preferably, the oxygen bridging the a- and 0-phosphates is replaced with another molecule that prevents formation of the phosphodiester bond. Particularly preferred derivatives comprise a substitution of the oxygen molecule bridging the a- and (3-phosphate with a carbon, nitrogen or sulfur molecule or a methylene group. Other nucleotide derivatives useful in the invention comprise a modification of the a-, (3- or y-phosphate group. The modification may comprise, for example, the substitution of a bridging or non-bridging oxygen molecule with a thiol, alkyl, carbonyl, amine, alcohol, aryl or an animo acid group.
[0013] Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. For example, preferred nucleotides include phosphate 5 esters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, and uridine.
[0014] Polymerases usefi.u.l in the invention include any nucleic acid polymerase capable of catalyzing a template-dependent addition of a nucleotide or nucleotide analog to a primer.
Depending on the characteristics of the target nucleic acid, a DNA polymerase, an RNA
polymerase, a reverse transcriptase, or a mutant or altered form of any of the foregoing can be used. According to one aspect of the invention, a thermophilic polymerase is used, such as ThermoSequenase , 9N:"', TherminatorTM, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffel fragment, VentTM and Deep VentTM DNA polymerase.

Brief Description of the Drawing [0015] Figure 1 is a schematic representation of a polymerization reaction that results in the addition of a nucleotide to a polynucleotide.
[0016] Figure 2 is a schematic representation of a DNA synthesis reaction performed in the presence of a nucleotide derivative.

Detailed Description [0017] The invention provides methods and compositions for improving the fidelity of a nucleic acid sequencing-by-synthesis reaction by reducing misincorporation of nucleotides.
While applicable to bulk sequencing methods, the invention is particularly useful in connection with single molecule sequencing methods. Methods of the invention improve the fidelity of DNA synthesis by blocking misincorporation of a nucleotide triphosphate in template/primer duplexes. According to the invention, when DNA synthesis reaction is performed in the presence of a nucleotide derivative that is complementary to the template nucleotide, the nucleotide derivative forms hydrogen bonds with the template nucleotide, but does not form a phosphodiester bond with the 3' hydroxyl group of the primer. Without incorporating into the primer, the nucleotide derivative is transiently held in place by the hydrogen bonds and blocks the misincorporation of the nucleotide triphosphate.
[0018] Figure 2 is a schematic representation of DNA synthesis reaction in which a nucleotide derivative (as shown a,(3-S-2'-deoxyadenosine 5'-triphosphate) (10) has formed hydrogen bonds with its complementary base (12) located on the template (14) opposite the incorporation site immediately adjacent the 3' terminus of the primer (16).
Because the nucleotide derivative lacks an oxygen molecule bridging the a and (3 phosphate groups, the polymerase (18) is not able to catalyze the formation of a phosphodiester bond between the nucleotide derivative (10) and the 3' hydroxyl group of the primer (16), but because the derivative (10) is blocking the incorporation site at the 3' terminus of the primer (16), the polymerase (18) can not misincorporate an incorrect (i.e., non-complementary) nucleotide(20).
[0019] According to the invention, a polymerization reaction is conducted in the presence of a polymerase, at least one labeled dNTP corresponding to a first nucleotide species, and at least one nucleotide derivative corresponding to a different nucleotide species. The nucleotide derivative comprises a modification that inhibits the formation of a phosphodiester bond between the derivative and the 3' hydroxyl group of a primer on a primer/template duplex.
If the template comprises a nucleotide opposite the incorporation site that is not complementary to the dNTP, but is complementary to the nucleotide derivative, the polymerase recognizes the nucleotide derivative as complementary to the template nucleotide and brings the derivative into close proximity with the template nucleotide, which facilitates the formation of hydrogen bonds between the derivative and its complementary nucleotide. With the nucleotide derivative blocking the incorporation site, misincorporation of the dNTP is prevented.
[0020] Methods and compositions of the invention are particularly well-suited for use in single molecule sequencing techniques. Substrate-bound primer/template duplexes are exposed to a polymerase, a labeled nucleotide corresponding to a first nucleotide species, and at least one nucleotide derivative corresponding to a different nucleotide species. The duplex may be simultaneously exposed to the polymerase, the labeled nucleotide, and the nucleotide derivative;
or it may be first exposed to the nucleotide derivative and then to the polymerase and labeled nucleotide. Typically, however, the duplex is simultaneously exposed to the polymerase, the labeled nucleotide, and three nucleotide derivatives, one corresponding to each of the three remaining nucleotide species. The duplexes are washed of unincorporated labeled nucleotides and nucleotide derivatives, and the incorporation of labeled nucleotide is determined. The identity of the nucleotide positioned on the template opposite the incorporate nucleotide is likewise determined. The polymerization reaction is serially repeated in the presence of a labeled nucleotide that corresponds to each of the other nucleotide species in order to compile a sequence of incorporated nucleotides that is representative of the complement to the template nucleic acid.
[0021] Certain non-limiting aspects of the invention are further described below in terms of general considerations and examples.

1. General Considerations A. Nrscleotides [0022] Nucleotides useful in the invention as free, unbound nucleotides for incorporation into a primer/template duplex include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. For example, preferred nucleotides include phosphate esters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, and uridine. Other nucleotides useful in the invention comprise an adenine, cytosine, guanine, thymine base, an xanthine or hypoxanthine; 5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4-methoxydeoxycytosine.
Also included are bases of polynucleotide mimetics, such as methylated nucleic acids, e.g., 2'-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation, and includes chain-terminating analogs. A
nucleotide corresponds to a specific nucleotide species if they share base-complementarity with respect to at least one base.
[0023] Nucleotides for nucleic acid sequencing according to the invention preferably comprise a detectable label. Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable. Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, Texas Red, green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3 dye, 5-(2'-aminoethyl)-aminonaphthalene-l-sulfonic acid (EDANS), BODIPY, 120 ALEXA or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid duplex to a digoxigenin, a fluorescein, an alkaline phosphatase or a peroxidase.

B. Nucleotide Derivatives [0024] Nucleotide derivatives useful in the invention include any nucleotide, including those described above in Section A, that forms hydrogen bonds with a complementary nucleotide on a template nucleic acid, but comprises a modification that prevents the formation of a phosphodiester bond with the 3' hydroxyl group of the primer. Preferred nucleotide derivatives will be recognized by the catalytic domain of the polymerase and brought in close proximity with its complementary nucleotide where hydrogen bonding can occur.
Accordingly, particularly useful nucleotide derivatives most closely resemble naturally-occurring substrates for polymerases in both chemical formula and structure. Thus, particularly useful nucleotide derivatives comprise phosphate esters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, and uridine. In a preferred embodiment, nucleotide derivatives comprise a triphosphate ester, such as dATP, dCTP, dGTP, dTTP or dUTP. A nucleotide derivative corresponds to a specific nucleotide species if they exhibit the same base-complementarity with respect to at least one base.
[0025] According to one feature, a nucleotide derivative comprises a modification of the oxygen bridging the a-phosphate and the 0-phosphate in a standard nucleotide triphosphate. For example, preferred modifications include the substitution of the oxygen molecule bridging the a-and (3-phosphate groups with a carbon, nitrogen or sulfur molecule or a methylene group. Other nucleotide derivatives useful in the invention comprise a modification of the a-, (3- or y-phosphate group, such as, for example, the substitution of a bridging or non-bridging oxygen molecule with a thiol, alkyl, carbonyl, amine, alcohol, aryl or an animo acid group; or a bulky group that physically interferes with polymerase function. Custom modified nucleotides are commercially available from, for example, TriLink BioTechnologies, Inc., San Diego, California, Alexis Biochemicals, Inc., Carlsbad, California and BIOLOG Life Science Institute, Germany.

C. Nucleic Acid Polymerases [0026] Nucleic acid polymerases generally useful in the invention include DNA
polymerases, RNA polymerases, reverse transcriptases, and mutant or altered forms of any of the foregoing. DNA polymerases and their properties are described in detail in, among other places, DNA Replication 2nd edition, Komberg and Baker, W. H. Freeman, New York, N.Y.
(1991).
Known conventional DNA polymerases useful in the invention include, but are not limited to, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1, Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et al., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli) DNA
polymerase (also referred to as VentTM DNA polymerase, Cariello et al., 1991, Polynucleotides Res, 19:
4193, New England Biolabs), 9 NmTM DNA polymerase (New England Biolabs), Stoffel fragment, ThermoSequenase (Amersham Pharmacia Biotech UK), TherminatorTM (New England Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J
Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien et al., 1976, J.
Bacteoriol, 127: 1550), DNA polymerase, Pyrococcus kodakaraensis KOD DNA
polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (from thermococcus sp. JDF-3, Patent application WO 0132887), Pyrococcus GB-D (PGB-D) DNA
polymerase (also referred as Deep VentTM DNA polymerase, Juncosa-Ginesta et al., 1994, Biotechniques, 16:820, New England Biolabs), UlTma DNA polymerase (from thermophile Thermotoga maritima; Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239; PE
Applied Biosystems), Tgo DNA polymerase (from thermococcus gorgonarius, Roche Molecular Biochemicals), E. coli DNA polymerase I (Lecomte and Doubleday, 1983, Polynucleotides Res.
11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J Biol. Chem. 256:3112), and archaeal DP11/DP2 DNA polymerase II (Cann et al., 1998, Proc Natl Acad. Sci. USA
95:14250-->5).
[0027] While mesophilic polymerases are contemplated by the invention, preferred polymerases are thermophilic. Thermophilic DNA polymerases include, but are not limited to, ThermoSequenase , 9 NmTM, TherminatorTM, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffel fragrnent, VentTM and Deep VentTM DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, and mutants, variants and derivatives thereof.
[0028] Reverse transcriptases useful in the invention include, but are not limited to, reverse transcriptases from HIV, HTLV-1, HTLV-II, FeLV, FIV, SIV, AMV, MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8 (1997); Verma, Biochim Biophys Acta.
473:1-38 (1977); Wu et al., CRC Crit Rev Biochem. 3:289-347(1975)).
[0029] Certain embodiments of the invention are described in the following examples, which are not meant to be limiting.

Exainple 1: Single Molecule Sequencing Using Nucleotide Derivatives [0030] The following nucleotide derivatives, each comprising a substitution of the 5 oxygen molecule bridging the a and 0 phosphate groups with a sulfur molecule, are prepared:
a,(3-S-2'-deoxyadenosine 5'-triphosphate; a,l3-S-2'-deoxycytidine 5'-triphosphate; a,(3-S-2'-deoxyguanosine 5'-triphosphate; and a,(3-S-2'-deoxythymidine 5'-triphosphate.
Primer/template duplexes are bound to a solid support in a concentration that results in individually optically resolvable duplexes. The bound duplexes are subjected to serial sequencing-by-synthesis cycles 10 in which a polymerase, labeled standard nucleotide , and nucleotide derivatives corresponding to each of the three non-standard nucleotide species are combined. The incorporation of a labeled nucleotide is determined, recorded and the reaction serially repeated with labeled nucleotide corresponding to each of the different nucleotide species and the appropriate nucleotide derivatives in order to compile a sequence that is representative of the complement of the target nucleic acid.

Exatnple 2: Assay for the Inhibition of Polymerase Activity [0031] A simple assay may be performed to assess the ability of a usefulness of nucleotide derivative in methods and compositions of the invention.
[0032] Primed DNA template is diluted into an appropriate volume of 20 mM Tris-HCI, pH 7.5 and the enzyme is diluted into an appropriate volume of 20 mM Tris-HCI, containing 2 mM (3-mercaptoethanol, and 100 mM KCI. 0.05 mg/mL primed DNA template and 0.01 U/ L
DNA polymerase are pipetted into microcentrifuge tubes or a 96-well plate in a 50 L reaction volume. 1.6 M nucleotide derivative is added to the test reactions. Blank reactions excluding enzyme and control reactions excluding test compound are also prepared using enzyme dilution buffer and test compound solvent, respectively. The reaction is initiated with reaction buffer with the following components 20 mM Tris-HCI, pH 7.5; 200 g/mL bovine serum albumin; 100 mM KCI; 2 mM (3-mercaptoethanol; 10 mM MgC12, 1.6 M dATP, dGTP, dCTP, and dTTP;
and a-33P-dAT. The reaction is incubated for sixty minutes at 37 C and then quenched by the addition of 20 L of 0.5M EDTA. 50 L of the quenched reaction is spotted onto Whatman DE81 filter disks and air dried. The filter disks are repeatedly washed with 150 mL 0.3M

ammonium formate, pH 8 until 1 mL of wash is <100 cpm. The disks are washed twice with 150 mL absolute ethanol and once with 150 mL anhydrous ether, dried and counted in 5 mL
scintillation fluid.
[0033] The percentage of inhibition of the test is calculated according to the following equation: % inhibition={1-(cpm in test reaction-cpm in blank)/(cpm in control reaction-cpm in blank)}X 100.
[0034] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.
Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (31)

1. A method for inhibiting nucleotide misincorporation in a nucleic acid synthesis reaction, the method comprising conducting a polymerization reaction on a nucleic acid duplex comprising a template and a primer in the presence of a nucleic acid polymerase, a first nucleotide corresponding to a first nucleotide species, and at least one nucleotide derivative corresponding to a different nucleotide species, wherein said nucleotide derivative comprises a modification that inhibits formation of a phosphodiester bond between the nucleotide derivative and a free 3' hydroxyl on a primer nucleotide.
2. The method of claim 1, further comprising removing nucleotide derivatives and nucleotides that are not incorporated into the primer.
3. The method of claim 2, further comprising the step of repeating said conducting and removing steps using a second nucleotide species and a nucleotide derivative corresponding to a nucleotide species different from said second species.
4. The method of claim 1, wherein said nucleotide derivative comprises a triphosphate group lacking an oxygen between an .alpha.-phosphate and a .beta.-phosphate such that said nucleotide derivative is prevented from forming a phosphodiester bond with said free 3' hydroxyl group on the primer.
5. The method of claim 4 wherein said derivative comprises substitution of an oxygen molecule with a carbon, nitrogen or sulfur molecule.
6. The method of claim 4 wherein said derivative comprises substitution of an oxygen molecule with a methylene group.
7. The method of claim 4 wherein said derivative comprises substitution of an oxygen molecule with a thiol, alkyl, carbonyl, amine, alcohol, aryl or an animo acid group.
8. The method of claim 1 wherein, if said template comprises a nucleotide opposite a position immediately adjacent to a 3' terminus of the primer that is complementary to said nucleotide derivative but not complementary to said first nucleotide, said nucleotide derivative forms a hydrogen bond with its complementary nucleotide so as to prevent said first nucleotide from bonding thereto and misincorporating into said primer in said nucleic acid synthesis reaction.
9. The method of claim 1 wherein said nucleic acid duplex is bound to a solid support.
10. The method of claim 1 comprising a plurality of said duplex, each member of said plurality being individually optically resolvable.
11. The method of claim 1 wherein said first nucleotide is detectably labeled.
12. A method for sequencing a nucleic acid, the method comprising the steps of:

(a) exposing a nucleic acid duplex comprising a template and a primer to a nucleic acid polymerase, a first labeled nucleotide , and at least one nucleotide derivative corresponding to a nucleotide species different from said first nucleotide;

wherein said nucleotide derivative comprises a modification that inhibits formation of a phosphodiester bond between the nucleotide derivative and a 3' hydroxyl group of the primer, and (b) removing nucleotide derivatives and labeled nucleotides that are not incorporated into the primer;

(c) determining incorporation of the labeled nucleotide and thereby identifying a nucleotide that is complementary to a nucleotide at a position on said template opposite the incorporated labeled nucleotide; and (d) repeating steps (a), (b) and (c) with a second labeled nucleotide and at least one nucleotide derivative corresponding to a nucleotide species different from said second labeled nucleotide.
13. The method of claim 12, wherein said nucleotide derivative comprises a triphosphate group having a molecule other than oxygen between an .alpha.-phosphate and a .beta.-phosphate.
14. The method of claim 13 wherein said derivative comprises substitution of an oxygen molecule with a carbon, nitrogen or sulfur molecule.
15. The method of claim 13 wherein said derivative comprises a substitution of an oxygen molecule with a methylene group.
16. The method of claim 2 wherein said nucleotide derivative comprises a triphosphate group having a modified .alpha.- or .beta.-phosphate.
17. The method of claim 16 wherein said modification comprises a substitution of an oxygen molecule with a thiol, alkyl, carbonyl, amine, alcohol, aryl or an animo acid group.
18. The method of claim 12 wherein said exposing step is performed in the presence of at least two nucleotide derivatives, each derivative corresponding to a nucleotide species different from said first nucleotide.
19. The method of claim 12 wherein said exposing step is performed in the presence of three nucleotide derivatives, each derivative corresponding to a nucleotide species different from said first nucleotide.
20. The method of claim 12, further comprising the step of compiling a sequence of nucleotides incorporated into said primer.
21. The method of claim 12 wherein said labeled nucleotide comprises a labeled dNTP.
22. The method of claim 12 wherein said first nucleotide is a deoxyadenosine triphosphate and said at least one nucleotide derivative corresponds to a nucleotide species selected from the group consisting of deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxyuridine triphosphate.
23. The method of claim 12 wherein said first nucleotide is a deoxyguanosine triphosphate thereof and said at least one nucleotide derivative corresponds to a nucleotide species selected from the group consisting of deoxyadenosine triphosphate, deoxycytidine triphosphate and deoxyuridine triphosphate.
24. The method of claim 12 wherein said first nucleotide is a deoxycytidine triphosphate thereof and said three nucleotide derivatives corresponds to the nucleotide species of deoxyadenosine triphosphate, deoxyguanosine triphosphate and deoxyuridine triphosphate.
25. The method of claim 12 wherein said first nucleotides is a deoxyuridine triphosphate thereof and said at least one nucleotide derivative corresponds to a nucleotide species selected from the group consisting of deoxyadenosine triphosphate, deoxyguanosine triphosphate and deoxycytidine triphosphate.
26. The method of claim 19 wherein said first nucleotide is a deoxyadenosine triphosphate thereof and said three nucleotide derivatives correspond to deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxyuridine triphosphate.
27. The method of claim 19 wherein said first nucleotide is a deoxyguanosine triphosphate and said three nucleotide derivatives correspond to deoxyadenosine triphosphate, deoxycytidine triphosphate and deoxyuridine triphosphate.
28. The method of claim 19 wherein said first nucleotide is a deoxycytidine triphosphate and said three nucleotide derivatives correspond to deoxyadenosine triphosphate, deoxyguanosine triphosphate and deoxyuridine triphosphate.
29. The method of claim 19 wherein said first nucleotide is a deoxyuridine triphosphate and said three nucleotide derivatives correspond to deoxyadenosine triphosphate, deoxyguanosine triphosphate and deoxycytidine triphosphate.
30. The method of claim 12, wherein said exposing step comprises first exposing said nucleic acid duplex to said at least one nucleotide derivative, then exposing said nucleic acid duplex to said nucleic acid polymerase and said labeled nucleotide.
31. A composition for use in a nucleic acid synthesis reaction on a primer/template duplex, the composition comprising:

a labeled nucleotide corresponding to a first nucleotide species, and at least one nucleotide derivative of a different nucleotide species, wherein said nucleotide derivative comprises a modification that prevents the formation of a phosphodiester bond between the nucleotide derivative and a 3' hydroxyl group of the primer.
CA002599477A 2005-01-28 2006-01-30 Methods and compositions for improving fidelity in a nucleic acid synthesis reaction Abandoned CA2599477A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11/046,448 US7482120B2 (en) 2005-01-28 2005-01-28 Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
US11/046,448 2005-01-28
PCT/US2006/004638 WO2006081583A2 (en) 2005-01-28 2006-01-30 Methods and compositions for improving fidelity in a nucleic acid synthesis reaction

Publications (1)

Publication Number Publication Date
CA2599477A1 true CA2599477A1 (en) 2006-08-03

Family

ID=36582013

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002599477A Abandoned CA2599477A1 (en) 2005-01-28 2006-01-30 Methods and compositions for improving fidelity in a nucleic acid synthesis reaction

Country Status (5)

Country Link
US (1) US7482120B2 (en)
EP (1) EP1848829B1 (en)
AT (1) ATE548466T1 (en)
CA (1) CA2599477A1 (en)
WO (1) WO2006081583A2 (en)

Families Citing this family (124)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE273381T1 (en) * 1997-02-12 2004-08-15 Eugene Y Chan METHOD FOR ANALYZING POLYMERS
AU8288101A (en) * 2000-07-07 2002-01-21 Visigen Biotechnologies Inc Real-time sequence determination
EP1354064A2 (en) 2000-12-01 2003-10-22 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US7668697B2 (en) * 2006-02-06 2010-02-23 Andrei Volkov Method for analyzing dynamic detectable events at the single molecule level
US7833709B2 (en) 2004-05-28 2010-11-16 Wafergen, Inc. Thermo-controllable chips for multiplex analyses
US20080076189A1 (en) * 2006-03-30 2008-03-27 Visigen Biotechnologies, Inc. Modified surfaces for the detection of biomolecules at the single molecule level
WO2008151023A2 (en) 2007-06-01 2008-12-11 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
CA2715385A1 (en) 2008-02-12 2009-08-20 Pacific Biosciences Of California, Inc. Compositions and methods for use in analytical reactions
US20090226906A1 (en) * 2008-03-05 2009-09-10 Helicos Biosciences Corporation Methods and compositions for reducing nucleotide impurities
US8632975B2 (en) 2009-06-05 2014-01-21 Life Technologies Corporation Nucleotide transient binding for sequencing methods
WO2011014811A1 (en) 2009-07-31 2011-02-03 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
ES2573277T3 (en) * 2009-08-14 2016-06-07 Epicentre Technologies Corporation Methods, compositions and kits for the generation of samples depleted in rRNA or for the isolation of rRNA from samples
US9890408B2 (en) 2009-10-15 2018-02-13 Ibis Biosciences, Inc. Multiple displacement amplification
US8470261B2 (en) 2010-05-06 2013-06-25 Ibis Biosciences, Inc. Integrated sample preparation systems and stabilized enzyme mixtures
EP2596127A2 (en) 2010-07-23 2013-05-29 Esoterix Genetic Laboratories, LLC Identification of differentially represented fetal or maternal genomic regions and uses thereof
CA2811294C (en) 2010-09-16 2016-06-28 Ibis Biosciences, Inc. Stabilization of ozone-labile fluorescent dyes by thiourea
CN105755545B (en) 2010-12-27 2019-05-03 艾比斯生物科学公司 The preparation method and composition of nucleic acid samples
WO2013022778A1 (en) 2011-08-05 2013-02-14 Ibis Biosciences, Inc. Nucleic acid sequencing by electrochemical detection
WO2013063308A1 (en) 2011-10-25 2013-05-02 University Of Massachusetts An enzymatic method to enrich for capped rna, kits for performing same, and compositions derived therefrom
EP3211100A1 (en) 2011-12-22 2017-08-30 Ibis Biosciences, Inc. Amplification primers and methods
EP3269820A1 (en) 2011-12-22 2018-01-17 Ibis Biosciences, Inc. Kit for the amplification of a sequence from a ribonucleic acid
WO2013096838A2 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids
WO2013096799A1 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples
US10150993B2 (en) 2011-12-22 2018-12-11 Ibis Biosciences, Inc. Macromolecule positioning by electrical potential
US9506113B2 (en) 2011-12-28 2016-11-29 Ibis Biosciences, Inc. Nucleic acid ligation systems and methods
US9803231B2 (en) 2011-12-29 2017-10-31 Ibis Biosciences, Inc. Macromolecule delivery to nanowells
JP2015503923A (en) 2012-01-09 2015-02-05 オスロ ウニヴェルスィテーツスィーケフース ハーエフOslo Universitetssykehus Hf Methods and biomarkers for the analysis of colorectal cancer
EP2802666B1 (en) 2012-01-13 2018-09-19 Data2Bio Genotyping by next-generation sequencing
EP2834370B1 (en) 2012-04-03 2019-01-02 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and crohn's disease
EP2844772B1 (en) 2012-05-02 2018-07-11 Ibis Biosciences, Inc. Dna sequencing
WO2013166302A1 (en) 2012-05-02 2013-11-07 Ibis Biosciences, Inc. Nucleic acid sequencing systems and methods
WO2014005076A2 (en) 2012-06-29 2014-01-03 The Regents Of The University Of Michigan Methods and biomarkers for detection of kidney disorders
EP3447150A1 (en) 2012-10-16 2019-02-27 Abbott Molecular Inc. Methods and apparatus to sequence a nucleic acid
WO2014152937A1 (en) 2013-03-14 2014-09-25 Ibis Biosciences, Inc. Nucleic acid control panels
US9701999B2 (en) 2013-03-14 2017-07-11 Abbott Molecular, Inc. Multiplex methylation-specific amplification systems and methods
US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
EP2971139A4 (en) 2013-03-15 2016-12-07 Abbott Molecular Inc Systems and methods for detection of genomic copy number changes
ES2716094T3 (en) 2013-03-15 2019-06-10 Ibis Biosciences Inc Methods to analyze contamination in DNA sequencing
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
ES2764096T3 (en) 2013-08-19 2020-06-02 Abbott Molecular Inc Next generation sequencing libraries
EP3083700B1 (en) 2013-12-17 2023-10-11 The Brigham and Women's Hospital, Inc. Detection of an antibody against a pathogen
WO2015107430A2 (en) 2014-01-16 2015-07-23 Oslo Universitetssykehus Hf Methods and biomarkers for detection and prognosis of cervical cancer
EP3151733B1 (en) 2014-06-06 2020-04-15 The Regents Of The University Of Michigan Compositions and methods for characterizing and diagnosing periodontal disease
EP3919621A1 (en) 2014-06-23 2021-12-08 The General Hospital Corporation Genomewide unbiased identification of dsbs evaluated by sequencing (guide-seq)
EP3161157A4 (en) 2014-06-24 2017-12-20 Bio-Rad Laboratories, Inc. Digital pcr barcoding
ES2864855T3 (en) 2014-07-24 2021-10-14 Abbott Molecular Inc Methods for the detection and analysis of mycobacterium tuberculosis
US10208339B2 (en) 2015-02-19 2019-02-19 Takara Bio Usa, Inc. Systems and methods for whole genome amplification
US10641772B2 (en) 2015-02-20 2020-05-05 Takara Bio Usa, Inc. Method for rapid accurate dispensing, visualization and analysis of single cells
AU2016250281A1 (en) 2015-04-15 2017-10-26 The General Hospital Corporation LNA-based mutant enrichment next-generation sequencing assays
US10392613B2 (en) 2015-07-14 2019-08-27 Abbott Molecular Inc. Purification of nucleic acids using copper-titanium oxides
WO2017011565A1 (en) 2015-07-14 2017-01-19 Abbott Molecular Inc. Compositions and methods for identifying drug resistant tuberculosis
US10077470B2 (en) 2015-07-21 2018-09-18 Omniome, Inc. Nucleic acid sequencing methods and systems
CN107849605B (en) 2015-07-21 2021-08-20 欧姆尼欧美公司 Nucleic acid sequencing methods and systems
AU2016319110B2 (en) 2015-09-11 2022-01-27 The General Hospital Corporation Full interrogation of nuclease DSBs and sequencing (FIND-seq)
US9850484B2 (en) 2015-09-30 2017-12-26 The General Hospital Corporation Comprehensive in vitro reporting of cleavage events by sequencing (Circle-seq)
CN108431223A (en) 2016-01-08 2018-08-21 生物辐射实验室股份有限公司 Multiple pearls under per drop resolution
US10626390B2 (en) 2016-02-08 2020-04-21 RGENE, Inc. Multiple ligase compositions, systems, and methods
WO2017160788A2 (en) 2016-03-14 2017-09-21 RGENE, Inc. HYPER-THERMOSTABLE LYSINE-MUTANT ssDNA/RNA LIGASES
US10982264B2 (en) 2016-04-22 2021-04-20 Omniome, Inc. Nucleic acid sequencing method and system employing enhanced detection of nucleotide-specific ternary complex formation
EP3449012B1 (en) 2016-04-29 2020-12-23 Omniome, Inc. Sequencing method employing ternary complex destabilization to identify cognate nucleotides
US10584379B2 (en) 2016-04-29 2020-03-10 Omniome, Inc. Method of nucleic acid sequence determination
US10597643B2 (en) 2016-04-29 2020-03-24 Omniome, Inc. Polymerases engineered to reduce nucleotide-independent DNA binding
WO2018013509A1 (en) 2016-07-11 2018-01-18 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for diagnosing and treating arrhythmias
EP3875603A1 (en) 2016-07-12 2021-09-08 Life Technologies Corporation Compositions and methods for detecting nucleic acid regions
WO2018017892A1 (en) 2016-07-21 2018-01-25 Takara Bio Usa, Inc. Multi-z imaging and dispensing with multi-well devices
WO2018034780A1 (en) 2016-08-15 2018-02-22 Omniome, Inc. Sequencing method for rapid identification and processing of cognate nucleotide pairs
CA3033741A1 (en) 2016-08-15 2018-02-22 Omniome, Inc. Method and system for sequencing nucleic acids
WO2018042251A1 (en) 2016-08-29 2018-03-08 Oslo Universitetssykehus Hf Chip-seq assays
EP3555290B1 (en) 2016-12-19 2022-11-02 Bio-Rad Laboratories, Inc. Droplet tagging contiguity preserved tagmented dna
CA3048415C (en) 2016-12-30 2023-02-28 Omniome, Inc. Method and system employing distinguishable polymerases for detecting ternary complexes and identifying cognate nucleotides
CA3049722C (en) 2017-01-10 2023-01-24 Omniome, Inc. Polymerases engineered to reduce nucleotide-independent dna binding
US10415029B2 (en) 2017-01-20 2019-09-17 Omniome, Inc. Allele-specific capture of nucleic acids
WO2018136487A1 (en) 2017-01-20 2018-07-26 Omniome, Inc. Process for cognate nucleotide detection in a nucleic acid sequencing workflow
CA3050852C (en) 2017-01-20 2021-03-09 Omniome, Inc. Genotyping by polymerase binding
DK3354746T3 (en) 2017-01-30 2019-09-02 Gmi Gregor Mendel Inst Fuer Molekulare Pflanzenbiologie Gmbh NEW SPIKE-IN NUCLEOTIDES FOR NORMALIZING SEQUENCE DATA
WO2018152162A1 (en) 2017-02-15 2018-08-23 Omniome, Inc. Distinguishing sequences by detecting polymerase dissociation
US9951385B1 (en) 2017-04-25 2018-04-24 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
US10161003B2 (en) 2017-04-25 2018-12-25 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
AU2018279728B2 (en) 2017-06-08 2023-07-20 The Brigham And Women's Hospital, Inc. Methods and compositions for identifying epitopes
WO2018236918A1 (en) 2017-06-20 2018-12-27 Bio-Rad Laboratories, Inc. Mda using bead oligonucleotide
WO2019002265A1 (en) 2017-06-26 2019-01-03 Universität Für Bodenkultur Wien Novel biomarkers for detecting senescent cells
CN111357054A (en) 2017-09-20 2020-06-30 夸登特健康公司 Methods and systems for differentiating between somatic and germline variations
AU2018353136B2 (en) 2017-10-19 2022-05-19 Pacific Biosciences Of California, Inc. Simultaneous background reduction and complex stabilization in binding assay workflows
EP3704247B1 (en) 2017-11-02 2023-01-04 Bio-Rad Laboratories, Inc. Transposase-based genomic analysis
CN111699253A (en) 2018-01-31 2020-09-22 生物辐射实验室股份有限公司 Methods and compositions for deconvolving a partitioned barcode
US11512002B2 (en) 2018-04-18 2022-11-29 University Of Virginia Patent Foundation Silica materials and methods of making thereof
EP4234718A3 (en) 2018-04-26 2023-11-29 Pacific Biosciences Of California, Inc. Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes
WO2019213619A1 (en) 2018-05-04 2019-11-07 Abbott Laboratories Hbv diagnostic, prognostic, and therapeutic methods and products
US11180794B2 (en) 2018-05-31 2021-11-23 Omniome, Inc. Methods and compositions for capping nucleic acids
WO2019231568A1 (en) 2018-05-31 2019-12-05 Omniome, Inc. Increased signal to noise in nucleic acid sequencing
US20190385700A1 (en) 2018-06-04 2019-12-19 Guardant Health, Inc. METHODS AND SYSTEMS FOR DETERMINING The CELLULAR ORIGIN OF CELL-FREE NUCLEIC ACIDS
CA3107983A1 (en) 2018-07-23 2020-01-30 Guardant Health, Inc. Methods and systems for adjusting tumor mutational burden by tumor fraction and coverage
AU2019312152A1 (en) 2018-07-24 2021-02-18 Pacific Biosciences Of California, Inc. Serial formation of ternary complex species
US11479816B2 (en) 2018-08-20 2022-10-25 Bio-Rad Laboratories, Inc. Nucleotide sequence generation by barcode bead-colocalization in partitions
JP2021536232A (en) 2018-08-30 2021-12-27 ガーダント ヘルス, インコーポレイテッド Methods and systems for detecting contamination between samples
WO2020047378A1 (en) 2018-08-31 2020-03-05 Guardant Health, Inc. Microsatellite instability detection in cell-free dna
US20200075124A1 (en) 2018-09-04 2020-03-05 Guardant Health, Inc. Methods and systems for detecting allelic imbalance in cell-free nucleic acid samples
DK3814533T3 (en) 2018-09-20 2021-11-15 Tamirna Gmbh Micro-RNA signatures to predict liver dysfunction
US20200131566A1 (en) 2018-10-31 2020-04-30 Guardant Health, Inc. Methods, compositions and systems for calibrating epigenetic partitioning assays
US20240011086A1 (en) 2018-11-15 2024-01-11 Omniome, Inc. Electronic detection of nucleic acid structure
US10710076B2 (en) 2018-12-04 2020-07-14 Omniome, Inc. Mixed-phase fluids for nucleic acid sequencing and other analytical assays
CN113454218A (en) 2018-12-20 2021-09-28 夸登特健康公司 Methods, compositions, and systems for improved recovery of nucleic acid molecules
US11041199B2 (en) 2018-12-20 2021-06-22 Omniome, Inc. Temperature control for analysis of nucleic acids and other analytes
AU2020216438A1 (en) 2019-01-31 2021-07-29 Guardant Health, Inc. Compositions and methods for isolating cell-free DNA
EP3924513B1 (en) 2019-02-14 2023-04-12 Pacific Biosciences of California, Inc. Mitigating adverse impacts of detection systems on nucleic acids and other biological analytes
WO2020176659A1 (en) 2019-02-27 2020-09-03 Guardant Health, Inc. Methods and systems for determining the cellular origin of cell-free dna
EP3976822A1 (en) 2019-05-31 2022-04-06 Guardant Health, Inc. Methods and systems for improving patient monitoring after surgery
EP4045683A1 (en) 2019-10-18 2022-08-24 Omniome, Inc. Methods and compositions for capping nucleic acids
DK3812472T3 (en) 2019-10-21 2023-02-20 Univ Freiburg Albert Ludwigs TRULY UNBIASED IN VITRO ASSAYS FOR PROFILING THE OFF-TARGET ACTIVITY OF ONE OR MORE TARGET-SPECIFIC PROGRAMMABLE NUCLEASES IN CELLS (ABNOBA-SEQ)
US20210214800A1 (en) 2019-11-26 2021-07-15 Guardant Health, Inc. Methods, compositions and systems for improving the binding of methylated polynucleotides
WO2021152586A1 (en) 2020-01-30 2021-08-05 Yeda Research And Development Co. Ltd. Methods of analyzing microbiome, immunoglobulin profile and physiological state
WO2021214766A1 (en) 2020-04-21 2021-10-28 Yeda Research And Development Co. Ltd. Methods of diagnosing viral infections and vaccines thereto
CA3177127A1 (en) 2020-04-30 2021-11-04 Guardant Health, Inc. Methods for sequence determination using partitioned nucleic acids
CN115836135A (en) 2020-05-05 2023-03-21 加利福尼亚太平洋生物科学股份有限公司 Compositions and methods for modifying polymerase-nucleic acid complexes
WO2021224677A1 (en) 2020-05-05 2021-11-11 Akershus Universitetssykehus Hf Compositions and methods for characterizing bowel cancer
US20220025468A1 (en) 2020-05-14 2022-01-27 Guardant Health, Inc. Homologous recombination repair deficiency detection
WO2023282916A1 (en) 2021-07-09 2023-01-12 Guardant Health, Inc. Methods of detecting genomic rearrangements using cell free nucleic acids
EP4189111A1 (en) 2020-07-30 2023-06-07 Guardant Health, Inc. Methods for isolating cell-free dna
JP2023540221A (en) 2020-08-25 2023-09-22 ガーダント ヘルス, インコーポレイテッド Methods and systems for predicting variant origin
EP4222279A1 (en) 2020-09-30 2023-08-09 Guardant Health, Inc. Methods and systems to improve the signal to noise ratio of dna methylation partitioning assays
WO2022140629A1 (en) 2020-12-23 2022-06-30 Guardant Health, Inc. Methods and systems for analyzing methylated polynucleotides
US20220411876A1 (en) 2021-03-05 2022-12-29 Guardant Health, Inc. Methods and related aspects for analyzing molecular response
EP4305200A1 (en) 2021-03-09 2024-01-17 Guardant Health, Inc. Detecting the presence of a tumor based on off-target polynucleotide sequencing data
US20230360725A1 (en) 2022-05-09 2023-11-09 Guardant Health, Inc. Detecting degradation based on strand bias

Family Cites Families (161)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
JPS5941169B2 (en) 1975-12-25 1984-10-05 シチズン時計株式会社 Elastomer
US4153855A (en) 1977-12-16 1979-05-08 The United States Of America As Represented By The Secretary Of The Army Method of making a plate having a pattern of microchannels
US4351760A (en) 1979-09-07 1982-09-28 Syva Company Novel alkyl substituted fluorescent compounds and polyamino acid conjugates
US4344064A (en) 1979-12-06 1982-08-10 Western Electric Co., Inc. Article carrying a distinctive mark
US4711955A (en) 1981-04-17 1987-12-08 Yale University Modified nucleotides and methods of preparing and using same
US5260433A (en) 1982-06-23 1993-11-09 Enzo Diagnostics, Inc. Saccharide specific binding system labeled nucleotides
US4707237A (en) 1982-09-09 1987-11-17 Ciba Corning Diagnostics Corp. System for identification of cells by electrophoresis
US5198540A (en) 1982-10-28 1993-03-30 Hubert Koster Process for the preparation of oligonucleotides in solution
US4994373A (en) 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
DE3329892A1 (en) 1983-08-18 1985-03-07 Köster, Hubert, Prof. Dr., 2000 Hamburg METHOD FOR PRODUCING OLIGONUCLEOTIDES
USRE34069E (en) 1983-08-18 1992-09-15 Biosyntech Gmbh Process for the preparation of oligonucleotides
US5360523A (en) 1984-03-29 1994-11-01 Li-Cor, Inc. DNA sequencing
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4865968A (en) 1985-04-01 1989-09-12 The Salk Institute For Biological Studies DNA sequencing
US4739044A (en) 1985-06-13 1988-04-19 Amgen Method for derivitization of polynucleotides
US4863849A (en) 1985-07-18 1989-09-05 New York Medical College Automatable process for sequencing nucleotide
US4757141A (en) 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof
US5242797A (en) 1986-03-21 1993-09-07 Myron J. Block Nucleic acid assay method
US4811218A (en) 1986-06-02 1989-03-07 Applied Biosystems, Inc. Real time scanning electrophoresis apparatus for DNA sequencing
US5242796A (en) 1986-07-02 1993-09-07 E. I. Du Pont De Nemours And Company Method, system and reagents for DNA sequencing
CA1340806C (en) 1986-07-02 1999-11-02 James Merrill Prober Method, system and reagents for dna sequencing
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
US4994372A (en) 1987-01-14 1991-02-19 President And Fellows Of Harvard College DNA sequencing
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US5202231A (en) 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
US6270961B1 (en) * 1987-04-01 2001-08-07 Hyseq, Inc. Methods and apparatus for DNA sequencing and DNA identification
US4994368A (en) 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays
US4942124A (en) 1987-08-11 1990-07-17 President And Fellows Of Harvard College Multiplex sequencing
US4793705A (en) 1987-10-07 1988-12-27 The United States Of America As Represented By The United States Department Of Energy Single molecule tracking
US4962037A (en) 1987-10-07 1990-10-09 United States Of America Method for rapid base sequencing in DNA and RNA
US4971903A (en) 1988-03-25 1990-11-20 Edward Hyman Pyrophosphate-based method and apparatus for sequencing nucleic acids
US4962020A (en) 1988-07-12 1990-10-09 President And Fellows Of Harvard College DNA sequencing
CH679555A5 (en) 1989-04-11 1992-03-13 Westonbridge Int Ltd
US4979824A (en) 1989-05-26 1990-12-25 Board Of Trustees Of The Leland Stanford Junior University High sensitivity fluorescent single particle and single molecule detection apparatus and method
US6346413B1 (en) * 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
US5424186A (en) 1989-06-07 1995-06-13 Affymax Technologies N.V. Very large scale immobilized polymer synthesis
US5800992A (en) * 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US5744101A (en) * 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5547839A (en) 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
US5112736A (en) 1989-06-14 1992-05-12 University Of Utah Dna sequencing using fluorescence background electroblotting membrane
ATE110142T1 (en) 1989-06-14 1994-09-15 Westonbridge Int Ltd MICROPUMP.
US5108892A (en) 1989-08-03 1992-04-28 Promega Corporation Method of using a taq dna polymerase without 5'-3'-exonuclease activity
US5096554A (en) 1989-08-07 1992-03-17 Applied Biosystems, Inc. Nucleic acid fractionation by counter-migration capillary electrophoresis
US5302509A (en) 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
CA2028261C (en) 1989-10-28 1995-01-17 Won Suck Yang Non-invasive method and apparatus for measuring blood glucose concentration
KR910012538A (en) 1989-12-27 1991-08-08 야마무라 가쯔미 Micro pump and its manufacturing method
US5091652A (en) 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
WO1991011533A1 (en) 1990-01-26 1991-08-08 E.I. Du Pont De Nemours And Company Method for isolating primer extension products from template-directed dna polymerase reactions
DE4006152A1 (en) 1990-02-27 1991-08-29 Fraunhofer Ges Forschung MICROMINIATURIZED PUMP
US5096388A (en) 1990-03-22 1992-03-17 The Charles Stark Draper Laboratory, Inc. Microfabricated pump
US5223414A (en) * 1990-05-07 1993-06-29 Sri International Process for nucleic acid hybridization and amplification
SE470347B (en) 1990-05-10 1994-01-31 Pharmacia Lkb Biotech Microstructure for fluid flow systems and process for manufacturing such a system
DE69106240T2 (en) 1990-07-02 1995-05-11 Seiko Epson Corp Micropump and method of making a micropump.
US5529679A (en) 1992-02-28 1996-06-25 Hitachi, Ltd. DNA detector and DNA detection method
US5888819A (en) * 1991-03-05 1999-03-30 Molecular Tool, Inc. Method for determining nucleotide identity through primer extension
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US5518900A (en) 1993-01-15 1996-05-21 Molecular Tool, Inc. Method for generating single-stranded DNA molecules
US5762876A (en) 1991-03-05 1998-06-09 Molecular Tool, Inc. Automatic genotype determination
US5167784A (en) 1991-09-04 1992-12-01 Xerox Corporation Sequencing large nucleic acid fragments
DE4135655A1 (en) 1991-09-11 1993-03-18 Fraunhofer Ges Forschung MICROMINIATURIZED, ELECTROSTATICALLY OPERATED DIAPHRAGM PUMP
US5265327A (en) 1991-09-13 1993-11-30 Faris Sadeg M Microchannel plate technology
US5405747A (en) 1991-09-25 1995-04-11 The Regents Of The University Of California Office Of Technology Transfer Method for rapid base sequencing in DNA and RNA with two base labeling
US5756285A (en) 1991-09-27 1998-05-26 Amersham Life Science, Inc. DNA cycle sequencing
US5674679A (en) 1991-09-27 1997-10-07 Amersham Life Science, Inc. DNA cycle sequencing
JP3509859B2 (en) * 1991-11-07 2004-03-22 ナノトロニクス,インコーポレイテッド Hybridization of chromophore and fluorophore conjugated polynucleotides to create donor-donor energy transfer systems
US5632957A (en) 1993-11-01 1997-05-27 Nanogen Molecular biological diagnostic systems including electrodes
US5209834A (en) 1992-03-09 1993-05-11 The United States Of America As Represented By The United States Department Of Energy Ordered transport and identification of particles
US5304487A (en) 1992-05-01 1994-04-19 Trustees Of The University Of Pennsylvania Fluid handling in mesoscale analytical devices
DE4220077A1 (en) 1992-06-19 1993-12-23 Bosch Gmbh Robert Micro-pump for delivery of gases - uses working chamber warmed by heating element and controlled by silicon wafer valves.
JPH0622798A (en) 1992-07-07 1994-02-01 Hitachi Ltd Method for determining base sequence
US5306403A (en) 1992-08-24 1994-04-26 Martin Marietta Energy Systems, Inc. Raman-based system for DNA sequencing-mapping and other separations
US5403709A (en) 1992-10-06 1995-04-04 Hybridon, Inc. Method for sequencing synthetic oligonucleotides containing non-phosphodiester internucleotide linkages
AU671820B2 (en) 1993-02-10 1996-09-12 Promega Corporation Non-radioactive DNA sequencing
US5436149A (en) 1993-02-19 1995-07-25 Barnes; Wayne M. Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension
US5547859A (en) 1993-08-02 1996-08-20 Goodman; Myron F. Chain-terminating nucleotides for DNA sequencing methods
US5659171A (en) 1993-09-22 1997-08-19 Northrop Grumman Corporation Micro-miniature diaphragm pump for the low pressure pumping of gases
US5470710A (en) * 1993-10-22 1995-11-28 University Of Utah Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
US5610287A (en) 1993-12-06 1997-03-11 Molecular Tool, Inc. Method for immobilizing nucleic acid molecules
CH689836A5 (en) 1994-01-14 1999-12-15 Westonbridge Int Ltd Micropump.
US5654419A (en) 1994-02-01 1997-08-05 The Regents Of The University Of California Fluorescent labels and their use in separations
US6028190A (en) * 1994-02-01 2000-02-22 The Regents Of The University Of California Probes labeled with energy transfer coupled dyes
US5631734A (en) 1994-02-10 1997-05-20 Affymetrix, Inc. Method and apparatus for detection of fluorescently labeled materials
EP0749497B1 (en) 1994-03-08 2000-09-06 Amersham Pharmacia Biotech UK Limited Modifying nucleotide analogues
US5763594A (en) 1994-09-02 1998-06-09 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US5872244A (en) * 1994-09-02 1999-02-16 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US6015668A (en) * 1994-09-30 2000-01-18 Life Technologies, Inc. Cloned DNA polymerases from thermotoga and mutants thereof
US5695934A (en) * 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5514256A (en) 1994-10-28 1996-05-07 Battelle Memorial Institute Apparatus for improved DNA sequencing
US5707506A (en) 1994-10-28 1998-01-13 Battelle Memorial Institute Channel plate for DNA sequencing
US5556790A (en) 1994-12-05 1996-09-17 Pettit; John W. Method for Automated DNA sequencing
US5786142A (en) 1995-05-30 1998-07-28 Visible Genetics Inc. Electrophoresis and fluorescence detection method
US5710628A (en) 1994-12-12 1998-01-20 Visible Genetics Inc. Automated electrophoresis and fluorescence detection apparatus and method
US5599695A (en) 1995-02-27 1997-02-04 Affymetrix, Inc. Printing molecular library arrays using deprotection agents solely in the vapor phase
US5876187A (en) * 1995-03-09 1999-03-02 University Of Washington Micropumps with fixed valves
US5795782A (en) * 1995-03-17 1998-08-18 President & Fellows Of Harvard College Characterization of individual polymer molecules based on monomer-interface interactions
US6362002B1 (en) * 1995-03-17 2002-03-26 President And Fellows Of Harvard College Characterization of individual polymer molecules based on monomer-interface interactions
US5750341A (en) 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5675155A (en) 1995-04-26 1997-10-07 Beckman Instruments, Inc. Multicapillary fluorescent detection system
US5885813A (en) * 1995-05-31 1999-03-23 Amersham Life Science, Inc. Thermostable DNA polymerases
US5861287A (en) * 1995-06-23 1999-01-19 Baylor College Of Medicine Alternative dye-labeled primers for automated DNA sequencing
US5856174A (en) * 1995-06-29 1999-01-05 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US5733729A (en) 1995-09-14 1998-03-31 Affymetrix, Inc. Computer-aided probability base calling for arrays of nucleic acid probes on chips
SE9504099D0 (en) * 1995-11-16 1995-11-16 Pharmacia Biotech Ab A method of sequencing
US5776767A (en) 1995-12-12 1998-07-07 Visible Genetics Inc. Virtual DNA sequencer
US5705018A (en) 1995-12-13 1998-01-06 Hartley; Frank T. Micromachined peristaltic pump
EP0866868B1 (en) 1995-12-15 2001-07-11 Amersham Life Science Inc Thermostable dna polymerase from thermoanaerobacter thermohydrosulfuricus and mutant enzymes thereof with exonuclease activity removed
US5945283A (en) * 1995-12-18 1999-08-31 Washington University Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer
US6361937B1 (en) * 1996-03-19 2002-03-26 Affymetrix, Incorporated Computer-aided nucleic acid sequencing
SE9601318D0 (en) * 1996-04-04 1996-04-04 Pharmacia Biosensor Ab Method for nucleic acid analysis
US6432634B1 (en) * 1996-04-18 2002-08-13 Visible Genetics Inc. Method, apparatus and kits for sequencing of nucleic acids using multiple dyes
WO1997043611A1 (en) * 1996-05-16 1997-11-20 Affymetrix, Inc. Systems and methods for detection of labeled materials
US6221654B1 (en) * 1996-09-25 2001-04-24 California Institute Of Technology Method and apparatus for analysis and sorting of polynucleotides based on size
US6020457A (en) * 1996-09-30 2000-02-01 Dendritech Inc. Disulfide-containing dendritic polymers
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US6024925A (en) * 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
US6017702A (en) * 1996-12-05 2000-01-25 The Perkin-Elmer Corporation Chain-termination type nucleic acid sequencing method including 2'-deoxyuridine-5'-triphosphate
US5876934A (en) * 1996-12-18 1999-03-02 Pharmacia Biotech Inc. DNA sequencing method
US6828094B2 (en) * 1996-12-20 2004-12-07 Roche Diagnostics Gmbh Method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules with the addition of a second thermostable DNA polymerase and its application
ATE273381T1 (en) * 1997-02-12 2004-08-15 Eugene Y Chan METHOD FOR ANALYZING POLYMERS
US6117634A (en) * 1997-03-05 2000-09-12 The Reagents Of The University Of Michigan Nucleic acid sequencing and mapping
US5837860A (en) * 1997-03-05 1998-11-17 Molecular Tool, Inc. Covalent attachment of nucleic acid molecules onto solid-phases via disulfide bonds
GB9716231D0 (en) * 1997-07-31 1997-10-08 Amersham Int Ltd Base analogues
US5882904A (en) * 1997-08-04 1999-03-16 Amersham Pharmacia Biotech Inc. Thermococcus barossii DNA polymerase mutants
US6346379B1 (en) * 1997-09-11 2002-02-12 F. Hoffman-La Roche Ag Thermostable DNA polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes
US6511803B1 (en) * 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US5976842A (en) * 1997-10-30 1999-11-02 Clontech Laboratories, Inc. Methods and compositions for use in high fidelity polymerase chain reaction
US6322968B1 (en) * 1997-11-21 2001-11-27 Orchid Biosciences, Inc. De novo or “universal” sequencing array
US6185030B1 (en) * 1998-03-20 2001-02-06 James W. Overbeck Wide field of view and high speed scanning microscopy
US20030022207A1 (en) * 1998-10-16 2003-01-30 Solexa, Ltd. Arrayed polynucleotides and their use in genome analysis
US6716394B2 (en) * 1998-08-11 2004-04-06 Caliper Technologies Corp. DNA sequencing using multiple fluorescent labels being distinguishable by their decay times
US6245507B1 (en) * 1998-08-18 2001-06-12 Orchid Biosciences, Inc. In-line complete hyperspectral fluorescent imaging of nucleic acid molecules
DE19844931C1 (en) * 1998-09-30 2000-06-15 Stefan Seeger Procedures for DNA or RNA sequencing
DE19849348A1 (en) * 1998-10-26 2000-04-27 Univ Ludwigs Albert Identification and/or sequencing of an unknown DNA or RNA sequence adjacent to a known DNA or RNA region comprises linker-mediated PCR following amplification by linear PCR
US6340750B1 (en) * 1998-12-18 2002-01-22 The Texas A&M University System Through bond energy transfer in fluorescent dyes for labelling biological molecules
US6361671B1 (en) * 1999-01-11 2002-03-26 The Regents Of The University Of California Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels
US6537755B1 (en) * 1999-03-25 2003-03-25 Radoje T. Drmanac Solution-based methods and materials for sequence analysis by hybridization
US6521428B1 (en) * 1999-04-21 2003-02-18 Genome Technologies, Llc Shot-gun sequencing and amplification without cloning
US7056661B2 (en) * 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6818395B1 (en) * 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6528258B1 (en) * 1999-09-03 2003-03-04 Lifebeam Technologies, Inc. Nucleic acid sequencing using an optically labeled pore
US6309836B1 (en) * 1999-10-05 2001-10-30 Marek Kwiatkowski Compounds for protecting hydroxyls and methods for their use
US6342326B1 (en) * 2000-05-10 2002-01-29 Beckman Coulter, Inc. Synthesis and use of acyl fluorides of cyanine dyes
US20030017461A1 (en) * 2000-07-11 2003-01-23 Aclara Biosciences, Inc. Tag cleavage for detection of nucleic acids
EP1354064A2 (en) * 2000-12-01 2003-10-22 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
WO2002055997A2 (en) * 2001-01-12 2002-07-18 Karolinska Innovations Ab Substrate for fluorescence analysis
US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis
US20030027140A1 (en) * 2001-03-30 2003-02-06 Jingyue Ju High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry
US6689478B2 (en) * 2001-06-21 2004-02-10 Corning Incorporated Polyanion/polycation multilayer film for DNA immobilization
US6613523B2 (en) * 2001-06-29 2003-09-02 Agilent Technologies, Inc. Method of DNA sequencing using cleavable tags
US6989267B2 (en) * 2001-07-02 2006-01-24 Agilent Technologies, Inc. Methods of making microarrays with substrate surfaces having covalently bound polyelectrolyte films
US6995841B2 (en) * 2001-08-28 2006-02-07 Rice University Pulsed-multiline excitation for color-blind fluorescence detection
WO2003020902A2 (en) * 2001-08-31 2003-03-13 Datascope Investment Corp. Methods for blocking nonspecific hybridizations of nucleic acid sequences
US6852492B2 (en) * 2001-09-24 2005-02-08 Intel Corporation Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication
US6982165B2 (en) * 2001-09-24 2006-01-03 Intel Corporation Nucleic acid sequencing by raman monitoring of molecular deconstruction
US20040054162A1 (en) * 2001-10-30 2004-03-18 Hanna Michelle M. Molecular detection systems utilizing reiterative oligonucleotide synthesis
US20040014096A1 (en) * 2002-04-12 2004-01-22 Stratagene Dual-labeled nucleotides
WO2004072294A2 (en) * 2003-02-12 2004-08-26 Genizon Svenska Ab Methods and means for nucleic acid sequencing
US8399196B2 (en) * 2003-02-21 2013-03-19 Geneform Technologies Limited Nucleic acid sequencing methods, kits and reagents

Also Published As

Publication number Publication date
US20060172313A1 (en) 2006-08-03
WO2006081583A2 (en) 2006-08-03
WO2006081583A3 (en) 2006-10-19
EP1848829A2 (en) 2007-10-31
EP1848829B1 (en) 2012-03-07
ATE548466T1 (en) 2012-03-15
US7482120B2 (en) 2009-01-27

Similar Documents

Publication Publication Date Title
EP1848829B1 (en) Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
US11396673B2 (en) Closed nucleic acid structures
US7682809B2 (en) Direct ATP release sequencing
AU2015246165B2 (en) Closed nucleic acid structures
US10752944B2 (en) Circularized templates for sequencing
JP6050820B2 (en) Use of G-clamp for improved allele-specific PCR
JP6409233B2 (en) Kits and methods
EP3601611B1 (en) Polynucleotide adapters and methods of use thereof
CN101671672A (en) Polyanion for improved nucleic acid amplification
Hu et al. Specificity enhancement of deoxyribonucleic acid polymerization for sensitive nucleic acid detection
US20080096765A1 (en) Methods and compositions for correcting misincorporation in a nucleic acid synthesis reaction
WO2010017228A1 (en) Nucleic acid sequence detection by void extension ligation amplification

Legal Events

Date Code Title Description
FZDE Discontinued